GlutaredoxinV1, Main, Exploration, bibRecord, 000F09

Stable and controllable RNA interference: Investigating the physiological function of glutathionylated actin.

Identifieur interne : 000F09 ( Main/Exploration ); précédent : 000F08; suivant : 000F10

Stable and controllable RNA interference: Investigating the physiological function of glutathionylated actin.

Auteurs : Jun Wang [États-Unis] ; Ephrem Tekle ; Hammou Oubrahim ; John J. Mieyal ; Earl R. Stadtman ; P Boon Chock

Source :

Proceedings of the National Academy of Sciences of the United States of America [ 0027-8424 ] ; 2003.

RBID : pubmed:12697895

Descripteurs français

English descriptors

KwdEn :
- 3T3 Cells (MeSH), Actins (metabolism), Animals (MeSH), Base Sequence (MeSH), Catalysis (MeSH), DNA, Complementary (MeSH), Glutathione (metabolism), Glutathione Reductase (metabolism), Mice (MeSH), Mice, Knockout (MeSH), Microscopy, Confocal (MeSH), Molecular Sequence Data (MeSH), RNA Interference (MeSH).
MESH :
- chemical , metabolism : Actins, Glutathione, Glutathione Reductase.
- 3T3 Cells, Animals, Base Sequence, Catalysis, DNA, Complementary, Mice, Mice, Knockout, Microscopy, Confocal, Molecular Sequence Data, RNA Interference.

Abstract

RNA interference is an effective method to silence specific gene expression. Its application to mammalian cells, however, has been hampered by various shortcomings. Recently, it was reported that introduction of 22-bp double-stranded RNAs (dsRNAs) would specifically suppress expression of endogenous and heterogeneous genes in various mammalian cell lines. However, using this method, we failed to knock out proteins of interest effectively. Here we report the development of a stable and controllable method for generating dsRNA intracellularly. Tetracycline-responsive transactivator-containing cells were transfected with a vector capable of tetracycline-induced bidirectionally overexpressing sense and antisense RNA to form dsRNA in vivo. With this method, glutaredoxin, monitored by Western blot, was knocked out by overexpressing 290-base sense and antisense RNA in NIH 3T3 cells controlled by tetracycline or doxycycline. By using these glutaredoxin knocked-out cells, we have demonstrated that actin deglutathionylation plays a key role in growth factor-mediated actin polymerization, translocalization, and reorganization near the cell periphery.

DOI: 10.1073/pnas.0931345100
PubMed: 12697895
PubMed Central: PMC154305

Affiliations:

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Le document en format XML

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<term>Base Sequence (MeSH)</term>
<term>Catalysis (MeSH)</term>
<term>DNA, Complementary (MeSH)</term>
<term>Glutathione (metabolism)</term>
<term>Glutathione Reductase (metabolism)</term>
<term>Mice (MeSH)</term>
<term>Mice, Knockout (MeSH)</term>
<term>Microscopy, Confocal (MeSH)</term>
<term>Molecular Sequence Data (MeSH)</term>
<term>RNA Interference (MeSH)</term>
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<keywords scheme="KwdFr" xml:lang="fr"><term>ADN complémentaire (MeSH)</term>
<term>Actines (métabolisme)</term>
<term>Animaux (MeSH)</term>
<term>Catalyse (MeSH)</term>
<term>Cellules 3T3 (MeSH)</term>
<term>Données de séquences moléculaires (MeSH)</term>
<term>Glutathion (métabolisme)</term>
<term>Glutathione reductase (métabolisme)</term>
<term>Interférence par ARN (MeSH)</term>
<term>Microscopie confocale (MeSH)</term>
<term>Souris (MeSH)</term>
<term>Souris knockout (MeSH)</term>
<term>Séquence nucléotidique (MeSH)</term>
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<term>Glutathione</term>
<term>Glutathione Reductase</term>
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<term>Glutathion</term>
<term>Glutathione reductase</term>
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<front><div type="abstract" xml:lang="en">RNA interference is an effective method to silence specific gene expression. Its application to mammalian cells, however, has been hampered by various shortcomings. Recently, it was reported that introduction of 22-bp double-stranded RNAs (dsRNAs) would specifically suppress expression of endogenous and heterogeneous genes in various mammalian cell lines. However, using this method, we failed to knock out proteins of interest effectively. Here we report the development of a stable and controllable method for generating dsRNA intracellularly. Tetracycline-responsive transactivator-containing cells were transfected with a vector capable of tetracycline-induced bidirectionally overexpressing sense and antisense RNA to form dsRNA in vivo. With this method, glutaredoxin, monitored by Western blot, was knocked out by overexpressing 290-base sense and antisense RNA in NIH 3T3 cells controlled by tetracycline or doxycycline. By using these glutaredoxin knocked-out cells, we have demonstrated that actin deglutathionylation plays a key role in growth factor-mediated actin polymerization, translocalization, and reorganization near the cell periphery.</div>
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Serveur d'exploration sur la glutarédoxine

Stable and controllable RNA interference: Investigating the physiological function of glutathionylated actin.

Stable and controllable RNA interference: Investigating the physiological function of glutathionylated actin.

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